PROJECT SUMMARY When it comes to tissue regeneration in vertebrates the newts stand out as the champions. These animals can regenerate many organs and body parts even as adults and of old age. Thus, the newts could provide much coveted answers that the field of regenerative biology and medicine seeks. Among all tissues that can be regenerated the lens is a classic case of transdifferentiation because when removed as a whole regeneration ensues from a different tissue, the iris. Interestingly only the dorsal side of the iris is capable for transdifferentiation and not the ventral even though they both are made up of pigment epithelial cells. Thus, there must exist fundamental differences between dorsal and ventral iris that allow regeneration from the one site and not the other. Via transcriptomic analysis we have identified interesting patterns of gene expression. Some of the genes that show high levels of expression in dorsal or ventral iris will be examined in this proposal. In particular, we will examine the role of highly-regulated transcriptional factors in dorsal or ventral iris in the induction process (specific aim 1). For example, we have identified Tbx5 and Vax2 as dorsal- and ventral ?specific respectively. Can the ventral iris be induced if we knock-down Vax2 or up-regulate Tbx5? Also we have seen interesting patterns of regulation of Ephrin-B2 and its receptor as well as of netrin-1 and its receptor UNC5 (specific aim 2). Ephrin-B2 is highly expressed in the dorsal iris, while netrin-1 in the ventral iris, with their receptors having opposite patterns. Since these pairs are involved in repulsion of cells we believe that they are involved in setting the boundaries between dorsal and ventral iris and disallow mingling of dorsal and ventral iris cells, thus ensuring correct regeneration. Moreover, it seems that Ephrin/netrin signaling is part of the Vax2/Tbx5 network, implying that all these genes might establish a novel pathway, which regulates lens regeneration. This will be investigated with gain- or loss-of-function experiments. Outcome of the proposed experiments will provide firm answers as to mechanisms of regeneration and will allow us to extend in other species with the ultimate goal to eventually induce lens regeneration in mammals.